Genomic Enhancers at 8q24 and Prostate Cancer The understanding of genetic predisposition to prostate cancer (PCa) and the identification of at-risk alleles have undergone a revolution during the past three years, mainly due to the utilization of high-throughput genomic technologies. For example, in a comprehensive multi-ethnic study of germline variation and PCa risk, we established multiple independent risk alleles in 3 regions that span ~500-kb on chromosome 8q24. The alleles were verified in many other subsequent studies. All the variants are located in non-protein coding sequences and are >200-kb from any known gene. A 5-Mb chromatin segment encompassing all the risk regions was profiled for RNA expression, histone modifications and locations occupied by RNA polymerase II and the androgen receptor. This led to the identification of transcriptional enhancers, which were verified using reporter assays. In two of them single nucleotide polymorphisms (SNPs) affected TCF7L2 and FoxA1 binding, respectively and in the latter androgen-dependent enhancer activity. Our overall hypothesis is: PCa predisposition at 8q24 is governed by SNPs in enhancers, which affect transcription factor binding and distant gene expression. In specific aim #1, the enhancers containing risk SNPs will be characterized in vitro with emphasis on how genetic variations affect the combinatorial regulation of enhancer activities (subaim 1.1). Special attention will be directed towards already-identified, TCF7L2-, FoxA1- and androgen receptor-mediated risk mechanisms. Additionally, in vivo enhancer activities will be investigated in mice carrying reporter genes controlled by candidate enhancer elements (subaim 1.2). The spatial and temporal pattern of reporter activity will disclose the cell types and developmental stages at which the risk SNP-containing enhancers are maximally operative and how these patterns are modified by the risk SNPs. In specific aim #2, we will identify the target genes of the enhancers. We intend to employ three approaches: (i) large bacterial artificial chromosomes to test alleles under different physiologic conditions in cell culture and in mice (subaim 2.1), (ii) chromatin looping assays to test candidate target genes (such as Myc) (submain 2.2) and (iii) an unbiased looping screen to identify novel target genes genome-wide (subaim 2.3). Successful completion of the aims will lead to a more complete understanding of the biological mechanisms underlying genetic associations with PCa risk, as observed with variants located in non-protein coding sequences at 8q24. Additionally, our approach may provide a novel paradigm to study many other genetic loci found in non-protein coding areas associated with other disease states.